Team:Bielefeld-Germany/Labjournal/August

From 2013.igem.org

The backbone and shipping vector psB1C3 was cut with EcoRI, SpeI, the insert ccmAH with EcoRI and XbaI to perfom a standard suffix insertion.
We used approx. 200ng insert and 42ng vector, which repressnts a 2-fold molar excess of insert.
The Ligation was incubated for 15 min at 37°C, followed by a heat-inactivation at 80°C for 20 min.
The subsequent gel electrophoresis did not yield any positiv results, probably due to the use of buffer and enzymes from two different manufactures.

A gel electrophoresis was performed to ensure that all former products have the correct size and this could be ruled out as an error source.
It showed that all products were indeed correct.

The backbone and shipping plasmid psB1C3 was amplified with the new designed insert-specific primers for mtrCAB. These primers have a specific overlap complementary to 20nt at the beginning and end of the insert. This was necessary, because we had to deal with constant religation of the vector. We investigated this issue and found out, that prefix and suffix of the psB1C3 vector have a very similarity, which facilates religation. The correct band was extracted from the gel and cleaned up.

NanoDrop:

4-0108-301: 1.1 ng/ul

4-0108-302: 1.0 ng/ul

We use a custom 15 ul reaction mixture, prepared in our lab. After thawing on ice, a total amount of 5 ul DNA is added and the whole mixture is incubated at 50°C for approx. 60min.

Mixture:

psB1C3 (4-0108-301): 2.2 ng

Fragment1 (4-2607-304): 13.1 ng

Fragment2 (4-2707-004): 16.9 ng

Fragment3 (4-2607-301): 14.4 ng

Notes: The inserts were not equimolar and not added in a 3-fold excess to the vector

Transformation to self-made electrocompetent E.coli cells. A total volume of 1ul could be used.